Somatic embryogenesis is a unique pathway for asexual propagation or somatic cloning in plants. The developmental process of somatic embryogenesis shares considerable similarity with that of zygotic embryogenesis (Zimmerman, 1993; Mordhorst et al., 1997) and this is likely due to the conservation in the underpinning cellular and molecular mechanisms between the two processes. Therefore, somatic embryogenesis provides an attractive model system for studying zygotic embryogenesis, particularly because zygotic embryos are encased by maternal tissues and difficult to access by biochemical and molecular tools. Moreover, in biotechnological applications, most economically important crop as well as non-crop plants are regenerated via somatic embryogenesis.
In contrast to organogenesis, which requires a high cytokinin to auxin ratio (Skoog and Miller, 1957; Sugiyama, 1999; Sugiyama, 2000), somatic embryogenesis does not require any external cytokinins, but rather is dependent on high concentrations of 2,4-D (Zimmerman, 1993; Mordhorst et al., 1997; Sugiyama, 2000), a synthetic chemical that has long been used as a functional analog of auxin. It is generally believed that somatic embryogenesis is mediated by a signaling cascade triggered by external auxin or 2,4-D (Zimmerman, 1993; Mordhorst et al., 1997; Schmidt et al., 1997). However, very little is known about the signal transduction pathway, particularly the molecular mechanism involved in the transition of a vegetative cell to an embryogenic competent cell.
During the last two decades, considerable efforts have been made to identify genes with altered expression patterns during somatic embryogenesis (Schmidt et al., 1997; Lin et al., 1996; Thomas, 1993). Most of these genes, however, are up-regulated only in late developmental stages, suggesting that they do not play a direct role in the vegetative-to-embryogenic transition. Thus far, the only exception is the carrot Somatic Embryogenesis Receptor-like Kinase (SERK) gene the expression of which appears to mark the vegetative-to-embryogenic transition; however, its function remains unclear (Schmidt et al., 1997).
An additional molecular approach was attempted by manipulating certain embryo-specific genes. The Arabidopsis Leafy cotyledon 1 (LEC1) gene, encoding a subunit of the HAP heterotrimeric transcription factor complex (HAP3), has been proposed as a key regulator for embryonic identity (Lotan et al., 1998). Mutations in the LEC1 locus result in defective embryo maturation as well as the conversion of cotyledons into true-leaf-like structures (Lotan et al., 1998; Meinke, 1992; Meinke et al., 1994). Constitutive overexpression of LEC1 leads to severely abnormal plant growth and development with occasional formation of somatic embryo-like structures (Lotan et al., 1998). The developmental fate of these embryo-like structures, however, remained unknown due to the lethality of LEC1 overexpression.
Another LEC gene, LEAFY COTYLEDON2 (LEC2), encodes a B3 domain transcription factor that acts primarily in developing seeds. Ectopic post-embryonic expression of LEC2 in transgenic plants induces the formation of somatic embryo-like and other organ-like structures and often confers embryonic characteristics to seedlings (Stone et al., 2001).
The Brassica napus BABY BOOM (BBM) gene encodes a member of the AP2/ERF transcription factor family, which is preferentially expressed during development of embryos and seeds (Boutilier et al., 2002). Ectopic expression of BBM in Arabidopsis and Brassia led to the formation of somatic embryo-like structures and cotyledon-like structures on seedlings (Boutilier et al., 2002). It should be pointed out that in all these case, it is not known whether the somatic-embryo-like structures can germinate into fertile plants.
Using a novel chemical-inducible activation tagging system Zuo et al (2002) identified the PGA6 gene, which can promote vegetative-to-embryogenic transition in Arabidopsis roots and other organs. DNA sequence analysis of the PGA6 gene identified it as corresponding to the WUSCHEL (WUS) gene (Endirzzi et al., 1996; Laux et al., 1996; Mayer et al., 1998). Significantly, somatic embryos formed by WUS/PGA6 over-expression can germinate into fertile plants upon inducer withdrawal (Zuo et al., 2002).
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the text and respectively grouped in the appended Bibliography.